Cardiac muscle contraction is dependent upon a cooperative interaction between thick and thin filament sarcomeric proteins. Tropomyosin (TM), an essential thin filament protein, interacts with actin and the troponin complex to regulate contractile activity. In vitro biochemical studies using either bacterially-expressed TM or purified myofibrillar TM preparations subject to various chemical modifications have implicated different biochemical and physiological functions to specific TM regions. Although the investigations have been informative, they are based on in vitro modifications and analyses and may not accurately reflect the biochemical and physiological dynamics which occur in the in vivo situation. The applicant's long-term objective is to understand the relative importance of TM in myofilament responsiveness during mechanical and biochemical activity of cardiac muscle. Using mutagenesis of myofilament proteins and transgenic mice as tools, the applicant will test the hypothesis that modulation of the cooperative process by which cardiac myofilaments are turned on and off is an important regulator of cardiac function. The Specific Aims address the following questions: (1) What is the functional impact of altered properties of TM isoforms, missense mutations, and phosphorylation on myofilament activity in vivo, in vitro, and in situ? (2) What are the changes in dynamics and level of activity of heart muscle contraction and relaxation associated with these alterations? And (3) What is the mechanism by which TM isoform switching and protein phosphorylation affect the cooperative activation of the myofilaments? The Specific Aims of this project are: (1) to examine and understand the biological function of the amino terminal, middle portion, and carboxy end of a-TM; and (2) to define the functional differences between the striated muscle isoforms of a- and b-TM. The selective modification of TM amino acids will provide in vivo information for understanding the role of TM in the mechanism of muscle contraction and in its interaction with actin and the toponin complex. The development of transgenic mouse models carrying defined genetic mutations provides an opportunity to assess TM function in the heart during both normal and diseased state.